Defining the sites of phosphorylation in specified proteins is frequently a necessary step for gaining insight into a given biological event. The most commonly employed technique for determining in-vivo phosphorylation sites involved the isolation of phosphopeptides from phosphoproteins that have been metabolically labeled with [32P] phosphate. Alternative MS approaches for identifying phosphopeptides are attractive because radioactive [32P] incorporation is not required and because MS can provide extremely rapid, accurate phosphopeptide identification. Nevertheless, the analysis of high MW phosphoproteins by MS still presents a formidable analytical challenge because of the large number of proteolytic peptides that have to be analyzed and because good coverage of the protein is required to ensure that all the phosphopeptides have been identified. In practice, not all of the expected peptides from a protein are observed in the mass spectrum and frequently phosph opeptides ions are suppressed compared to non-phosphorylated peptides. Therefore, a method to specifically purify and identify phosphopeptides would be extremely valuable. We have developed a new approach to phosphopeptide isolation and phosphorylation mapping based upon specific chemical modification of phospho-Ser/Thr residues. Because phospho-Ser/Thr amino acids are labile under alkaline conditions, phosphoserine residues are cont... readily converted to S-ethylthiolcysteine by b-elimination and subsequent 1,2-ethanedithiol addition. We then attached a biotin moiety via a short linker to the free thiol group. Thus, we have essentially replaced a biotin moiety via a short linker to the free thiol group. Thus, we have essentially replaced the phosphate groups on protein by biotin moieties. After enzymatic digestion of the converted phosphoprotein, we use an avidin column to enrich the converted phosphopeptides. The high affinity of avidin for biotin allows vigorous washing in order to remove non-specifically interactive peptides. The combination of this chemical modification and mass spectrometry facilities ready identification of phosphorylation sites.